The present invention relates to compositions and methods for reducing mortality of cultured marine and freshwater (crustacean) animals due to viral infections. More specifically, the invention relates to compositions and methods for inducing tolerance against infections caused by viruses such as the White Spot Syndrome Virus in crustaceans.
As the demand for food throughout the world increases, a great deal of effort has been expended finding ways to more efficiently produce food, both animal and vegetable, to satisfy the demand. Sea life, including crustaceans and fish, has long been a source of high quality protein for human consumption. However, the activities of the wild marine product industries have, in recent years, been severely restricted because of environmental contamination problems and overfishing. Fish catches have become much smaller and it has been difficult to keep fishing grounds productive.
Attempts have been made to grow monocultures of aquatic animals (e.g., shrimp farming) under varying levels of controlled conditions. Often such farms provide a large proportion of a particular kind of seafood consumed. For example, approximately half of the penaeid shrimp consumed in the United States in 1993-94 were from farms. Aquaculture systems of the prior art (or mariculture systems, for sea-born animals) are either open (i.e., water is constantly replenished from an outside source) or closed (i.e., the same water is recirculated through the system).
Successful mariculture has been undertaken mainly in coastal areas using estuarine or coastal waters which are rich in nutrients provided by effluents from the land. Efficient production of crustaceans, fish, and shells have been undertaken by surrounding part of a marine area such as a calm gulf, a lake or an estuarine river having favorable conditions with nets, or by building ponds on land which take advantage of the tidal flow from the sea or the natural flow of water from rivers or estuaries. Large shrimp farms have thus been built in the coastal zones of Latin American and Southeast Asian countries. These shrimp culture systems rely partially on the eco-systems and marine food chains that develop in the breeding ponds to supply the feed for the shrimp. In certain cases, natural foods produced in ponds are supplemented by shrimp feed or the natural food chains are stimulated by the addition of fertilizer.
Unfortunately, shrimp and other crustaceans are susceptible to infection by a variety of viral and bacterial pathogens, such as parvoviruses, baculoviruses, Vibrio, and necrotizing hepatopancreatitis bacterium. The lack of an immune response of Crustacea to viral disease is well documented (Arala-Chaves and Sequeira (2000)), and viral infections result in significantly reduced yields of the cultured animals. In particular, one such pathogen, the White Spot Syndrom Virus (WSSV), poses a serious threat to shrimp farming industries. The WSSV is particularly virulent, the resulting infection has a high mortality rate, and the virus a wide range of potential hosts, including several species of penaid shrimp, crabs, and freshwater prawns. For instance, Flegel and Alday-Sanz (1998) documented the presence of WSSV in thirty-three different species of Crustacea in Asia. Moreover, crabs and planktonic shrimps which are common inhabitants of shrimp ponds can serve as asymptomatic carriers.
WSSV possibly originated in China in 1993, subsequently spread to the rest of Asia, and reached the eastern United States and the Western Hemisphere in 1995-1996. With few exceptions, the epidemic resulted in catastrophic mortality of shrimp, spreading rapidly among shrimp farms in every affected area. Later, in early 1999, it was detected in Central America and shortly thereafter in Ecuador with similar effects. The events of the past few years have shown that semi-intensive shrimp culture systems that dominate the industry in the Western Hemisphere, in particular those based on Penaeus vannamei, are highly vulnerable to the epizootic WSSV infection. The negative commercial consequences are by far the most serious threat that has ever confronted the industry, and the only measures available to minimize expected losses are those of prevention, including, e.g., virus screening of broodstock and larvae, and widespread utilization of semiclosed or closed cultivation (see, e.g., U.S. Pat. Nos. 5,947,057 and 5,732,654).
Curiously, as noted by Pasharawipas et al. (1997) and Flegel and Pasharawipas (1998), the intensity of many viral epidemics, including monodon baculovirus (MBV; which targets the hepatopancreas), immune hepatopancreatic and hematopoietic necrotic virus (IHHNV), yellow-head virus (YHV) and WSSV, have a tendency to diminish in intensity in roughly two to three years. Mechanisms such as genetic selection for resistance in the host shrimp and attenuation of the virus did not provide a plausible explanation for this phenomenon in view of the relatively short time available for these mechanisms to develop in nature. Instead, several other observations pointed to another mechanism being at work. First, the presence of innocuous viral infections was noted in many histological samples of animals with active viral infections. Several instances of viral infection without an inflammation in the tissues characterizing lethal infection are documented in shrimp and other Crustacea. Second, ample evidence of endemic IHHNV in P. monodon without inflammatory and lethal infection suggested a means by which a population might coexist with a virus over a long period of time. This coexistence might then allow genetic selection in the host and attenuation of virus might result in long term accommodation between the two organisms. Third, animals previously exposed to virus seemed to resist infection whereas xe2x80x9cnaivexe2x80x9d animals without a history of previous exposure where highly susceptible to lethal infections when challenged by YHV. A similar response was observed when virus-free P. stylirostris were exposed to apparently healthy P. stylirostris that had been reared previously in the presence of the virus. The xe2x80x9cexperiencedxe2x80x9d animals were shown to be carriers of an active viral infection that proved to be lethal to the xe2x80x9cnaxc3xafvexe2x80x9d animals.
These observations led the authors to present the xe2x80x9ctolerancexe2x80x9d theory (Pasharawipas et al., 1997) and later, the xe2x80x9caccommodationxe2x80x9d theory (Flegel and Pasharawipas, 1998), on how lethal infections are converted to innocuous infections. According to the accommodation theory, tolerance to viral infections in crustaceans is the manifestation of an active system for accommodation that is based on membrane binding involving specific memory, leading to suppression of viral triggered apoptosis and to persistent innocuous infections. It was then suggested that exposure of young larval stages to viral particles or sub-unit viral particles could result in innocuous infections rather than mortality once the animals are exposed to virus. The authors termed such a hypothetical composition xe2x80x9ctolerinexe2x80x9d as opposed to xe2x80x9cvaccinexe2x80x9d, since it was theorized that these compositions would produce their effect through different mechanisms than those of vaccines.
However, so far, no such tolerine or tolerine-like composition has been produced or any beneficial effect demonstrated in a laboratory bioassay using a live virus challenge. Neither has a tolerine product applicable to routine hatchery rearing of shrimp post-larvae, or methods of successfully applying such a tolerine composition to induce protection or tolerance against WSSV infection in cultured shrimp, been presented. Thus, there is a need for new compositions and methods for reducing the impact of WSSV infection in cultured shrimp and other crustaceans. The invention addresses these and other needs in the art.
The present invention provides compositions and methods for inducing tolerance to White Spot Syndrome Virus (WSSV) in crustaceans.
The invention also provides a tolerine composition comprising inactivated WSSV. Preferably, the WSSV of the composition has been chemically inactivated, or, for example, treated at a low pH such as pH 5.6 to inactivate the virus. In one embodiment of the invention, the tolerine composition further comprises a dispersing agent and/or a preservative.
The invention also provides a method for inducing tolerance to WSSV infection in shrimps, comprising exposing the shrimp larvae to a tolerine composition. In a preferred embodiment, the shrimp larvae are substantially at the Z-1 developmental stage. In another preferred embodiment, shrimp larvae are exposed to the tolerine for at least about 30 minutes, preferably for at least about 45 minutes. In yet another preferred embodiment, the tolerine composition is admixed with shrimps in an aqueous solution. Preferably, a total of about 1 liter of tolerine is added to about 10 to about 100 liters, more preferably about 20 to about 80 liters, and even more preferably about 50 liters of shrimp solution.